Abstract

Cytochromes P450 CYP3A5 and CYP3A4 exhibit differential plasticity that underlies differences in drug metabolism and drug-drug interactions. To extend previous studies, CYP3A4 and CYP3A5 were cocrystallized with clotrimazole, a compact ligand that binds to the heme iron in the catalytic center of the active site. Binding studies indicate that clotrimazole exhibits tight binding to CYP3A5 with a binding affinity (Kd) of <0.01 μM like that of CYP3A4. A single clotrimazole is bound to the heme iron in CYP3A4 that triggers expansion of active site cavity that reflects a loss of aromatic interactions between phenylalanine sidechains in the distal active site and increased conformational entropy for the F-F’ connector due to reorientation of Phe-304 to accommodate clotrimazole. In contrast to CYP3A4, the CYP3A5 Phe-304 exhibits an induced fit along with Phe-213 to form edge-to-face aromatic interactions with heme-bound clotrimazole. These aromatic interactions between aromatic amino acids propagate by induced fits with a second clotrimazole residing in the distal active site and a third clotrimazole bound in an expanded entrance channel as well as between the three clotrimazoles. The large, expanded entrance channel surrounded by the C-terminal loop and the F’ and A’ helices in CYP3A5 suggests conformational selection for the binding of clotrimazole due to its large girth, which may also cause the entrance channel to remain open after the binding of the first clotrimazole to the heme iron. The additional binding sites suggest a path for sequential binding of one molecule to reach and bind to the heme iron.